Changes in pathological phenotype of C9orf72 ALS iPSC-derived lines after treatment with Morpholino oligomers
M. BERSANI
1, M. TAIANA
1,, F. BIELLA
1, M. NIZZARDO
2, S. GHEZZI
2, N. BRESOLIN
2, G.P. COMI
2, S. CORTI
21 Department of Pathophysiology and Transplants, University of Milan 2 IRCCS Ca’ Granda Foundation, Ospedale Maggiore Policlinico Milano
Amyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by progressive degeneration of motor neurons (MNs). GGGGCC repeat expansions in C9ORF72 gene are the most common identified genetic cause, and even if their pathogenic processes are still unknown, many possible mechanisms have been proposed, including loss of function of the C9Orf72 protein, gain of function from accumulation of RNA foci and sequestration of RNA binding proteins (RBPs), and toxicity caused by dipeptide repeats proteins (DPRs) produced by repeat-associated non-ATG (RAN) translation.
One promising and reliable method to understand C9-ALS pathogenesis is represented by patient-specific induced pluripotent stem cells (iPSC)-derived lines and iPSC-derived MNs. Our therapeutic approaches include the use of antisense oligonucleotides (ASOs) designed to bind complementary mRNA and interfere with specific biological processes. In our laboratory, two different ASOs with Morpholino chemistry have been designed: against the C9ORF72 expansion motif and against the whole C9ORF72 gene; our aim is to characterize the pathological phenotype of the C9-ALS iPSC-derived lines and evaluate the therapeutic effect of ASOs administration on specific pathological markers. We reprogrammed iPSCs from C9-ALS patients and controls and differentiated them into MNs using a 14-days protocol. We investigated the phenotype of the C9-ALS lines compared to controls, evaluating cells survival, pluripotency and motor neuronal markers, R-loops formation, STMN2 expression, defects in axonal elongation. Next step was transfecting ALS-MNs with different Morpholinos and evaluating modification of the previously mentioned pathological markers. Interestingly, we identified in C9-ALS iPSC-derived lines pathological features such as accumulation DNA damage, R-loops increase, and minor axonal elongation, with decreased levels of Nfh, Stmn1 and Sept7 genes. After Morpholino treatments, we observed that ASO therapy could partially rescue the pathological phenotype. Our results suggest that patient specific iPSCs and iPSC-derived MNs are a valuable tool to deepen the knowledge of C9ORF72 pathogenic mechanisms, and that Morpholino-mediated approaches represent a very promising therapeutic strategy that needs to be further validated.
ABSTRACT C9 iPSCs-DERIVED MNs CHARACTERIZATION
Morpholinos (MOs) are antisense oligonucleotides (ASOs) with phosphorothioate-ribose backbone replaced with phosphorodiamidate-linked morpholino backbone that is refractory to metabolic degradation. They are properly designed to bind complementary mRNA and determine an interfering with specific biological processes.
MOA has been designed to bind C9ORF72 promoter and silence C9 gene, while MOB binds C9 GGGGCC expansion. Both MOA and B treatments induce a decrease of DNA damage response activation.
MORPHOLINO OLIGOMERS
PSCs and iPSCs-derived MNs present some pathological hallmarks of C9ORF72-ALS, proving to be an useful patient specific model to study pathogenic mechansim of the disease and to test the efficacy of new therapies. We plan to expand C9 iPSCs-MNs derived characterization and validate the obtained read-outs on new lines.. Our preliminary data showed promising therapeutic potential for MOA and MOB. We will deepen our evaluation assessing the effects of their administration on the pathological features in mature C9 MNs.
CONCLUSIONS
This work is funded by Regione Lombardia (TRANS-ALS project) to GPC and Ministry of Health to GPC.
AKNOWLEDGEMENTS
Fig.1: Differentiation of iPSCs lines into spinal MNs using a multistep protocol (Mauri et al, 2015)
AXONAL DEVELOPMENT GENES AXONAL LENGHT
AXONAL ELONGATION
CTRL C9
DNA DAMAGE
CTRL PZ1 CTRL PZ2 C9 PZ1 C9 PZ2
Fig.3:C9 iPSC-derived MNs showed significant impairment of axonal elongation
Fig. 4: 9 iPSC-derived MNs presented R-loops increase compared to controls
Fig. 2: C9 iPSC-derived MNs show an increase of DNA damage marker phospho-H2AX and CDKN1A
Fig.5: C9 MNs show a strong decrease in axonal-
related genes STMN1, STMN2, SEPT7 and NfH levels in qPCR
MOLECULAR THERAPY: MORPHOLINO
R-LOOPS
AXONAL DEVELOPMENT GENES
C9 CTRL
Fig.6:
Mos rescue axonal elongation in C9 MNs
Fig.7: qPCR analysis confirms that MO treatment increases the levels of STMN-2 and NfH in C9 derived lines
h2ax
h2ax
C9 C9+MOA C9+MOB
Smi32 Smi32 Smi32
STMN2
DNA DAMAGE
C9 C9+MOA C9+MOB
Fig.8: immunostaining quantification analysis for h2ax confirms that MO treatment decreases expression of DNA damage proteins in iPSCs-derived MNs
R-LOOPS
Fig.9: MOB reduced significantly R-
loops formation in C9 iPSCs lines
h2ax h2ax h2ax
C9 C9+MOB
***
***
STMN2
0.000 0.001 0.002 0.003 0.004
C9
C9+MOA
C9+MOB 0.000000
0.000005 0.000010 0.000015 0.000020 0.000025
